Pharmacokinetics and Pharmacogenomics of Ribociclib in Black Patients with Metastatic Breast Cancer: The LEANORA study

Abstract Underrepresented populations' participation in clinical trials remains limited, and the potential impact of genomic variants on drug metabolism remains elusive. This study aimed to assess the pharmacokinetics (PK) and pharmacogenomics (PGx) of ribociclib in self-identified Black women with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2) advanced breast cancer. LEANORA (NCT04657679) was a prospective, observational, multicenter cohort study involving 14 Black women. PK and PGx were evaluated using tandem mass spectrometry and PharmacoScan™ microarray (including CYP3A5*3 , *6 , and *7 ). CYP3A5 phenotypes varied among participants: 7 poor metabolizers (PM), 6 intermediate metabolizers (IM), and one normal metabolizer (NM). The area-under-the-curve did not significantly differ between PMs (39,230 hr*ng/mL) and IM/NMs (43,546 hr*ng/mL; p = 0.38). The incidence of adverse events (AEs) was also similar. We found no association between CYP3A5 genotype and ribociclib exposure. Continued efforts are needed to include diverse populations in clinical trials to ensure equitable treatment outcomes.


Introduction
Breast cancers can be subclassi ed based on receptors on tumor cells; these play a critical role in tumor biology and determining the optimal treatment approach for each patient 1 .Hormone (estrogen and/or progesterone) receptor-positive (HR+) and human epidermal growth factor receptor 2 negative (HER2-) breast cancer accounts for approximately 70% of all breast cancers 1 .The cornerstone of treatment for patients with endocrine-sensitive advanced HR+/HER2-breast cancer is endocrine therapy (ET), which consists of selective estrogen receptor modulators (SERM: tamoxifen), aromatase inhibitors (AIs: letrozole, anastrozole, exemestane) or selective estrogen receptor degraders (SERDs: fulvestrant) 2,3 .However, patients often develop resistance to these treatments, and progression of disease.Novel agents have been added to ET with the goal of improving patient outcomes.
Ribociclib is an active drug that is metabolized (into inactive metabolites) by CYP3A 12 .Therefore, the recommended ribociclib dose varies with CYP3A activity.Prior studies identi ed that ribociclib is metabolized by CYP3A, and concomitant use of strong CYP3A inhibitors increased ribociclib exposure by 3.2-fold 13 .The FDA recommends the use of alternative therapy or a 50% dose reduction (i.e., from 600 mg to 400 mg) if ribociclib is used with strong CYP3A inhibitors 14 .However, it is unknown if dose changes are needed based on variations in baseline CYP3A activity, which is constituted by CYP3A4 and CYP3A5.
Variations in the genes encoding these proteins, CYP3A4 and CYP3A5, could mimic the effect of inhibition of these enzymes.There is currently insu cient evidence regarding the potential in uence of CYP3A4 and CYP3A5 genotype on ribociclib exposure. 15For CYP3A4, outside of rare variants (e.g., CYP3A4*22, CYP3A4*6), there is a paucity of evidence describing the broad impacts of CYP3A4 variants on enzymatic function.In contrast, variations in CYP3A5 genotype are well established with tacrolimus dosing and pharmacokinetic properties 16 .In brief, patients who express CYP3A5 (i.e., intermediate or normal metabolizers) are more likely to experience subtherapeutic trough levels and require higher doses of tacrolimus.Interestingly, previous evidence, including the FDA label, suggests that African American patients require higher doses of tacrolimus than White patients [16][17][18] .Although race and genetic ancestry are distinct, these ndings align with the fact that patients of African ancestry are likely (~ 85%) to be expressors (normal or intermediate metabolizers) of CYP3A5 16,17,19 .This is contrary to patients of European ancestry where ~ 85% are CYP3A5 poor metabolizers (CYP3A5 non-expressors) 16 .CDK4/6i were approved based on large studies.However, there was limited representation of racial and ethnic minorities; for example, in the pivotal trials studying ribociclib, only 41 of 2,066 (< 2%) patients identi ed as Black 6-8 .This is primarily a concern because the lack of underrepresented patients re ects a potential continuation of disparities in care 20 .Secondly, it also suggests ribociclib dosing was tested in a population that predominantly lacks CYP3A5 expression (poor metabolizers), and other patient populations may require a different dose to achieve similar outcomes in safety and e cacy.Speci cally, patients who self-identify as Black or African American were underrepresented in ribociclib studies and there may be differences in ribociclib metabolism and ribociclib exposure based on CYP3A5 genotype, which could lead to altered clinical outcomes.LEANORA was a prospective study that assessed the pharmacogenomics (PGx) and pharmacokinetics (PK) of ribociclib in self-identi ed Black patients with advanced breast cancer.

Patient population
Between May 2021 and March 2024, 84 patients were reviewed; 63 patients were ineligible (e.g., not metastatic disease, EKG abnormality) or declined to participate, three participants withdrew after providing consent (i.e., participant changed their mind, inability to obtain blood samples for analyses [n = 2]), 3 participants enrolled in the non-Hispanic White (NHW) cohort that closed to due low accrual (Supplemental gure S1).Results, including the NHW cohort, are available in supplemental tables S1-S4.
Fourteen self-identi ed Black participants with HR+/HER2-advanced breast cancer completed the study, and their data was included in this analysis.Demographic characteristics are described in Table 1.The median age was 61.5 years, and none of the patients identi ed as Hispanic.The most common sites of metastatic disease were bone, soft tissue, and lung.All patients received ribociclib and ET (letrozole or fulvestrant) per standard of care.The majority (13) of the patients received letrozole, while one received fulvestrant.Three patients received concurrent ovarian suppression.

Human Liver Microsome Analysis
Ribociclib metabolism was evaluated in human liver microsomes obtained from three individuals not from this current study, each harboring a different CYP3A5 genotype: CYP3A5*1/*1 (NM), CYP3A5*1/*3 (IM), and CYP3A5*3/*3 (no CYP3A5 function or PM).Consistent with the importance of CYP3A5 in ribociclib metabolism, the formation of major ribociclib metabolites was correlated with CYP3A5 genotype status (Fig. 1).Microsomes from a CYP3A5 NM had the greatest ribociclib metabolite formation followed by IM and PM.

Pharmacokinetic and Pharmacogenetic Findings
The primary endpoint, area under the curve (AUC

Toxicity pro le
Adverse events (AEs) of interest are summarized in Table 3. Overall, the toxicity pro le was consistent with previous reports with ribociclib.The most common toxicities were leukopenia, nausea, vomiting, and diarrhea.The reported grade 3 toxicities included neutropenia, nausea, vomiting, transaminitis, and increased creatinine.This study was not powered to assess differences in AEs between PMs and IM/NMs.There was a similar number of all grade toxicities by CYP3A5 phenotype: 100% (7 of 7) vs. 71% (5 of 7) for IM/NM vs. PMs, respectively (p = 0.46).PMs also had a similar number of grade 3 + AEs (29%, 2/7) compared to IM/NMs (29%, 2/7) (p = 1).Table 4 shows a similar change in laboratory values (i.e., transaminases, absolute neutrophil count [ANC]) and QTc by CYP3A5 phenotype between baseline and mid-cycle and end of cycle 1.Supplemental table S3 provides data about the toxicity pro le in the entire study, and supplemental table S4 provides additional AE data related to QTc prolongation, including one case from cohort 2 where a participant experienced grade 1 QTc prolongation and was unknowingly prescribed a prohibited medication that could prolong the QTc (venlafaxine).Univariate analyses of clinical factors did not identify signi cant associations with ribociclib AUC (Table 5).These ndings should be considered with caution due to the small sample size and because the study was not powered to assess these differences.variants and three different genotypes in 104 variants.This exploratory analysis of 14 participants identi ed one variant with a p < 0.05 for ribociclib AUC TAU : CYP3A4 rs2246709 (p = 0.0041), with A/G being associated with elevated AUC TAU compared to A/A (Fig. 2A and 2B).

Discussion
In the LEANORA trial, we found no association between CYP3A5 genotype and ribociclib exposure or adverse events among self-identi ed Black participants.Our ndings do not suggest that CYP3A5 screening is warranted to determine the safety of ribociclib.However, correlative studies of larger trials using ribociclib may provide more insight into interindividual exposure and response to ribociclib.This was seen in the microsome data in which metabolites from a CYP3A5 NM had the greatest ribociclib metabolite formation (inactive) followed by IM and PM, respectively.
These ndings are aligned with prior population pharmacokinetics (popPK) and pharmacokinetic/pharmacodynamic (PK/PD) models that found ribociclib pharmacokinetics are not affected by sex or ECOG performance status 21 .We identi ed weak associations between age and increased ribociclib AUC TAU and weight and decreased AUC TAU .This small inverse correlation between body weight and ribociclib exposure is aligned with data included in the FDA review of ribociclib but is not expected to be clinically relevant 22 .Similar to ribociclib, palbociclib is predominantly metabolized by CYP3A 15 .A case report identi ed that CYP3A5 expression (CYP3A5*1/*3, IM) was associated with lower ribociclib plasma concentration 23 .James et. al. conducted several studies that identi ed ~ 63% of ribociclib metabolism is produced by CYP3A 24 .There is signi cant overlap in the substrates of CYP3A4 and CYP3A5, and it is possible that both impact drug metabolism 13,24 .However, this current study's ndings in participants with cancer suggest CYP3A5 genotype has a minimal role.The recombinant human enzyme studies suggest CYP3A4 is the primary enzyme responsible for ribociclib metabolism 24 .
It is possible that genetic variations in CYP3A4 may be associated with ribociclib pharmacokinetics, but variations in CYP3A4 are less common.One pre-identi ed CYP3A4 variant of interest, CYP3A4*22, was present in one participant in this trial, which limited further investigation of that speci c variant.Additionally, the exploratory candidate gene analysis identi ed one variant of CYP3A4, rs2246709, potentially associated with ribociclib AUC TAU , which happened to be located in CYP3A4.However, this is an intronic variant with no obvious impact on enzyme expression or function.A query in the National Institutes of Health (NIH) LDLink did not identify linkage disequilibrium with other variants (highest r 2 was 0.36). 25Future steps could include exploring the impact of rare variants on ribociclib exposure in larger samples or may also explore genetic associations (e.g., KCNH2, SCN5A, SNTA1) with QTc prolongation secondary to ribociclib therapy 26 .
This study was focused on identifying a potential association between CYP3A5 genotype and ribociclib PKs.Therefore, concomitant medications were strictly regulated.In the one case where a patient was unknowingly prescribed a prohibited medication (venlafaxine), administration of this agent was associated with an adverse event (Grade 1 QTc prolongation).The ET was limited to letrozole, fulvestrant with or without ovarian function suppression, given potential interactions of other agents, such as tamoxifen and anastrozole, which could have impacted the PK studies 27 .
In terms of ribociclib dosing, the current recommended dose for patients with mBC is 600 mg daily for 21 days, followed by 7-day off treatment.There are prespeci ed dose reductions based on tolerance.An analysis of the MONALEESA 2, 3, and 7 studies revealed that 42% of patients required a dose reduction, which did not affect the e cacy of the drug 28 .The AMALEE (NCT03822468) study compared the standard 600 mg dose of ribociclib to 400 mg with the same schedule; after a median follow-up of 14 months, the study did not meet its primary endpoint of non-inferiority for the low dose based on objective response rate 29 .However, the study showed a favorable toxicity pro le of the lower dose, particularly for neutropenia and QTc prolongation.The approval of cancer-directed therapies has traditionally been based on the maximum tolerated dose, and in recent years, there has been a shift to consider optimal biological doses.The latter is not based on toxicities but rather on other cancer-speci c endpoints such as AMALEE 30 .We encourage researchers to rethink study endpoints to improve patient outcomes while limiting toxicity when possible.
Although no difference was found in ribociclib pharmacokinetics by CYP3A5 genotype, this trial has important implications for clinical practice.It is reassuring for patients and clinicians that the standard of care is not affected by the CYP3A5 genotype.Additionally, this trial represents the largest known cohort of ribociclib PK data in patients who self-identify as Black.
It is critical to understand the differences between race and ancestry when conducting and interpreting research.Race and ethnicity are social constructs that can be self-ascribed and based on shared physical or social qualities 31 .They do not necessarily re ect genetic ancestry; therefore, using race or ethnicity as proxies for generic ancestry can be inaccurate 19 .Genetic ancestry refers to people in the past to whom an individual is biologically connected 32,33 .Genetic ancestry and genealogy can in uence frequencies of genetic variants (e.g., carrier of HLA-B*15:02 or CYP3A5*1).Providing clinical recommendations based on genetic testing in pharmacology remains controversial as the impact of testing on clinical outcomes remains unknown [33][34][35] .It is likely that race, ethnicity, and genetic ancestry all contribute to patient outcomes.This trial studied an underrepresented population based on race as well as genetic variants more prevalent among those of African ancestry.
that could impact CYP3A, other proteins related to ribociclib pharmacokinetics, or risk of adverse events (Supplemental tables S5-S6).
Participants were considered postmenopausal if: i) they have had prior bilateral oophorectomy; ii) age ≥ 60 years; iii) age < 60 years and have had amenorrhea for 12 or more months (in the absence of chemotherapy, tamoxifen, toremifene, or ovarian suppression) and follicle-stimulating hormone (FSH) and estradiol in the postmenopausal range per local normal ranges.
The study initially enrolled participants into two independently powered cohorts based on self-identi ed race: cohort 1) African American or Black participants and cohort 2) NHW participants.However, the planned cohort 2 only enrolled three patients, and due to di culty enrolling patients, this cohort was closed.In this manuscript, we present the data about cohort 1.However, the results of the patients enrolled in cohort 2 are described in the supplemental material.

Human Liver Microsome Analysis
Human Liver Microsomes (20 pmol/mL CYP/reaction; 37°C, Seksui Xenotech, Kansas City, KS) were added to preincubated reaction buffer containing 0.5M potassium phosphate buffer (pH 7.4), NADPH solutions A and B according to the manufacturer's instructions (Corning, Corning NY), and ribociclib (Selleckchem, Houston, TX) or tacrolimus (MedChemExpress, Monmouth Junction, NJ) dissolved in acetonitrile at a nal concentration of 25µm (0.1% acetonitrile).Aliquots (100 µL) were removed from the reaction immediately after the addition of HLMs (0min) and 60 minutes later.Reactions were terminated by the addition of 100 µL of ice-cold acetonitrile followed by 1 minute of vortexing.Tubes were then centrifuged at maximum speed for 10 minutes at 4°C and harvested supernatant was stored (-20°C) until pharmacokinetic analysis.Bioanalytical measurements of ribociclib and metabolite concentrations were made using an LC-MS/MS assay described in the next section.

Pharmacokinetic and Pharmacogenetic Testing
Serial blood samples were collected for PK and PGx analyses (Fig. 3).The FDA label reports that ribociclib has an elimination half-life of 32 hours and a maximum plasma concentration (C max ) of 1 to 4 hours 14 .For sampling to occur at steady-state concentrations, the collection day was scheduled between days 8 and 16 of therapy during cycle 1.Blood samples were collected immediately prior to the ribociclib dose, and 0.5hr ± 5min, 1hr ± 5min, 2hr ± 15min, 4hr ± 15min, and 6hr ± 15min after the daily dose of ribociclib, processed to plasma and stored at -80 C until bioanalysis.
For in vitro metabolism experiments and clinical PK plasma samples, ribociclib total concentrations (i.e. protein bound + unbound) were measured using a validated and robust LC-MS/MS assay using palbociclib as an internal standard.Brie y, samples were mixed with acetonitrile containing palbociclib as an internal standard and run alongside calibration and quality control (QC) standards, ranging from 0.25-50 µM.The analyte (ribociclib) and internal standard were chromatographically separated using a Phenomenex Polar Omega C18 column (5 µm, 2.1x100 mm) on a Shimadzu Prominence HPLC system (Shimadzu, Columbia, MD).A gradient mobile phase consisting of 0.1% formic acid (aq) and 0.1% formic acid in acetonitrile was owed at a rate of 0.4 mL/min; the injection volume was 5 µL.Following chromatographic separation and elution, the compounds were detected using tandem mass spectrometric detection in the positive ion mode.MRM transitions were m/z 435.

Statistical analysis
The area under the curve (AUC tau ) between CYP3A5 PMs vs. IM/NMs was compared with the exact Wilcoxon rank-sum test due to small sample sizes, with alpha set to 0.05.Secondly, a multiple regression analysis was intended to identify variables associated with ribociclib AUC.Covariates to be assessed include CYP3A5 metabolism status, CYP3A4 metabolism status, renal function, liver function, age, race, weight, sex, use of "medication not recommended", AI, number of ribociclib doses taken.However, the multiple linear regression was not conducted due to the inability to provide valid inference with the small sample size.Fisher's exact test assessed the AEs and grade 3 + AEs to day 28.Descriptive statistics were used to characterize the data pro le, frequency, and percentages for categorical variables and mean (SD) or median [IQR] for continuous variables based on the data normality.Laboratory and electrocardiographic assessments were done per the current standard of care at baseline, close to C1D15, and at the end of the rst cycle 14 .The QT interval was corrected using the Bazett method.Analysis was performed using SAS™ software (v 9.1; Cary, NC, USA) and R Statistical Software (v4.3.1;R Core Team 2023).

Exploratory Candidate Gene Analysis
We conducted an exploratory candidate gene analysis among measured variants in genes that were previously linked to ribociclib pharmacokinetics.The objective of this analysis was to assess if there was a strong genetic effect on ribociclib AUC TAU .A literature review in May 2024 (search terms "ribociclib AND pharmacokinetics" in PubMed) identi ed 80 articles.Articles were screened for content related to genes or proteins related to ribociclib metabolism.Seventeen articles met inclusion and led to the identi cation of 15 genes of interest: ABCB1, ABCG2, CYP1A2, CYP2C19, CYP2C9, CYP3A4, CYP3A5, FMO1, FMO3, NR1/2, PPARα, POR, SLCO1B1, SLCO1B3, SULT2A1.Genetic data were available for 14 of 15 genes, with it unavailable for PPARα.The list of variants interrogated is available in supplemental table S7.The association of each of the variants with ribociclib AUC TAU was evaluated by an exact

Figure 2 P
Figure 2

Table 1
TAU ), was similar between CYP3A5 PM (39,230 hr*ng/mL; IQR: 18,745 to 57,566 hr*ng/mL) vs. IM/NM (43,546 hr*ng/mL; IQR: 35,298 to 46,647 hr*ng/mL; p = 0.38).PK properties by CYP3A5 phenotype are summarized in Table2.Similarly, there were no statistical differences in maximum concentration (C max ) or in the time to reach C max suggesting that differences in CYP3A5 genotype did not impact ribociclib exposure.
*Median and interquartile range are shown for continuous data.Abbreviations: AUC: area under the curve, C max : maximum concentration, IM/NM: intermediate metabolizers/normal metabolizers; PM: poor metabolizers; T max : time to reach maximum concentration

Table 3
Genetic data were available for all 14 participants in the exploratory candidate gene analysis.Out of 495 variants assessed, 285 variants were identical among all participants.No further evaluation was conducted among those 285 variants.The remaining 210 variants yielded two different genotypes in 106 The list of speci c types of AEs has been abbreviated.A complete list of recorded adverse events are available in supplemental table S3.Abbreviations: AE: adverse events; IM/NM: intermediate metabolizers/normal metabolizers; PM: poor a Spearman's correlation and the corresponding p-value b 0: fully functional, 1: limited functional c an exact Wilcoxon rank sum test-based p-value d an exact Jonckheere-Terpstra trend test-based p-value e the number of unrecommended medications taken by each participant.The two participants taking one unrecommended medication were prescribed a medication that may prolong QT interval (mirtazapine, ondansetron).The participant with two unrecommended medications was prescribed metformin (ribociclib may increase metformin levels) throughout the study and atorvastatin (CYP3A substrate) was paused the day before ribociclib initiation until D13 after completion of sample collection for PK analysis.Abbreviations: AUC: area under the curve Exploratory candidate gene analysis 3 ◊ 322.2 and m/z 448.4 ◊ 380.2 for ribociclib and palbociclib, respectively.Additionally, ribociclib metabolites were also detection and quantitated based off the parent ribociclib calibration: m/z 421.3 ◊ 322.2 (Demethyl); m/z 407.3 ◊ 322.2 (Di-demethyl); m/z 451.3 ◊ 338.3 (Hydroxyl); m/z 449.3 ◊ 336.3 (Oxidation).NM vs. PM.A second sample size calculation identi ed this cohort would provide 86.5%power to detect a 2 vs 1 difference in AUC pending the SD is 0.56.Alpha set at 0.05.The cohort that enrolled self-identi ed non-Hispanic White participants was terminated prematurely due to low accrual (n = 3).A buccal swab for CYP3A5 genotyping (Kailos Genetics) was obtained during the screening period for each patient to ensure adequate sample sizes of each phenotype per cohort.
were translated to phenotypes via the Hardy-Weinberg Eq. 16.Based on known frequencies for African Americans (normal metabolism: 35%; intermediate metabolism: 48%; poor metabolism: 16%), a sample size of 18 would provide 80% power at an α = 0.05 to detect a 2-fold change in AUC (SD is 0.56) between CYP3A5 PM vs. CYP3A5 expressors (i.e., CYP3A5 IM/NM).After the enrollment of 14 participants who self-identi ed as African American or Black, the distribution of CYP3A5 results was a 1:1 split for CYP3A5 IM/